2016 Fiscal Year Annual Research Report
| Project/Area Number |
16F16089
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| Research Institution | National Institute for Basic Biology |
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Principal Investigator |
藤森 俊彦 基礎生物学研究所, 初期発生研究部門, 教授 (80301274)
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| Co-Investigator(Kenkyū-buntansha) |
DAY TIMOTHY 基礎生物学研究所, 初期発生研究部門, 外国人特別研究員
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| Project Period (FY) |
2016-04-22 – 2018-03-31
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| Keywords | 鉄イオン |
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| Outline of Annual Research Achievements |
I investigated the morphological changes of both the uterus and the extraembryonic tissues through analysis of 3D rendered images of post-implantation embryos. Proof of principle was determined by making 3D reconstructions of heart tissues from serially sectioned 9.5 dpc embryos, scanned at high resolution, aligned by unique and precise automated software, and then delineated from surrounding tissues by manual tracing throughout 241 serial sections using the free open-source software FIJI/TrakEM2. Subsequently 21 implanted blastocysts from 4.5 dpc to 6.5 dpc have been traced and digital reconstructions have been created.
Gene expression analysis with reverse-transcription quantitative PCR of iron regulatory genes STEAP1-4, lactotransferrin, SLC11a2, and SLC40a1 in uteri of all estrus cycles and post-implantation decidual tissues found differential expression. As expression of SLC40a1 had highest correlation with uterine receptivity, I proceeded to test gene expression through serial section immunofluorescence using paraffin-embedded sections of uteri both non-pregnant and at stages of 4.5-9.5 days. SLC40a1 protein was upregulated in non-pregnant females is particularly high in the luminal epithelium and in the glandular epithelium of the uterus. During implantation expression is high, but is gradually lost in the surrounding decidua and LE of the blastocyst implantation site, with evident SLC40a1+ puncta formation after in the peripheral glandular epithelium from 6.5 dpc, suggesting downregulation of expression in the luminal epithelium through internalization.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
As expression of SlC40a1 was confirmed in the uterus before and after implantation, and due to its importance as the only known exporter of iron from mammalian cells, to further understand its role in supporting the development of the embryo, import of SLC40a1 (C57BL/6N-Slc40a1<tm1a(EUCOMM)Hmgu>/H) conditional knockout mouse sperm was recently completed and the mouse line is being rederived to in-house facilities through in vitro fertilization. The mouse will be used as both a straight knockout and with conditional knockout specifically in the uterus.
Continuation of characterization of SLC40a1 during embryo development in the uterus through antibody staining is currently ongoing. For increased optimization of SLC40a1 tissue specific staining, immunohistochemistry utilizing DAB peroxidase staining is also being performed. Concurrently, colocalization analysis of SLC40a1 with LAMP1, EEA1, and detection of hepcidin, an endogenous negative regulator of SLC40a1 protein expression, is currently underway to investigate SLC40a1's expression loss.
To understand the nature of iron presence and availability during implantation and development of the mouse embryo, immunofluorescence staining using an anti-mouse ferritin antibody is being performed. Ferritin protein is an indicator of iron presence, as increased iron intake into the cell triggers ferritin expression. Preliminary results indicate increased expression of ferritin and thus increased iron in the visceral endoderm and surrounding supporting extra-embryonic tissues.
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| Strategy for Future Research Activity |
After successful rederivation of the targeted conditional knockout mouse SLC40a1, it will be crossed to two transgenic mouse cre lines for specific deletions in the adult female mouse; Wnt7a-cre to remove SLC40a1 expression in the luminal epithelium of the uterus, and progesterone receptor-cre for a general deletion in the whole uterus. In parallel, expression of flippase will result in a null-reporter allele, where cells expressing endogenous SLC40a1 will be marked by β-galactosidase expression. Expression of β-galactosidase will be used to verify immunofluorescence and rt-qPCR data. Subsequent fertility and implantation in these uteri will be assessed.
Iron status through use of potassium ferrocyanide and ferricyanide (Perls' Prussian Blue) staining revealed below detection threshold levels of iron presence at implantation sites, although detectable in control tissues. For a more sensitive approach to detect even minute iron presence, electron microscopy analysis for the presence of iron in paraffin-embedded wild-type uteri and embryonic tissues will be performed. Subsequently, mutant uteri and wild-type embryos implanted therein will also be assessed by this sensitive method. If maternal iron regulatory genes expressed in the uterus can control fetal growth by regulating iron availability, I expect to find decreased iron presence in the developing embryo, loss of growth and patterning defects.
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Research Products
(2 results)
